A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using fluorescence instead of, or in addition to, reflection and absorption. The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. The sample can either be fluorescing in its natural form (like chlorophyll) or it may be treated with a fluorescing stain.
A basic fluorescence microscope that is well known in the art includes a light source and several filters that correspond to a wavelength matching a fluorescence stain. An excitation filter is provided for selecting an excitation wavelength of light from the light source, and a dichroic beamsplitter reflects light from the light source to illuminate the specimen. The illuminated light is separated from the much weaker emitted fluorescence with an emission filter. The fluorescing areas can be observed in the microscope and shine out against a dark background with high contrast.
Fluorescence microscopes use a special lighting system to view a specimen that has been treated with a fluorescent stain. The lighting system requires a special light source that, amongst other things, outputs a high level of light at certain key wavelengths that correlate to peak excitation wavelengths of corresponding fluorescent stains. The light source must be very powerful since the vast majority of the light needs to be filtered out to produce a nearly monochromatic, dichromatic, or trichromatic source. Most manufacturers currently use either mercury or xenon light sources, or in some cases, metal halide sources. Regardless of the type of light source used, the sources typically have a power rating of about 50 to 200 watts.
The most commonly used light sources are mercury and xenon arc type lamps. These arc type lamps require an expensive power supply to obtain a stable arc and produce a stable image of the specimen. The arc type lamps also have a relatively short life (about 200 hours) that requires laboratories to change and realign the lamp bulb frequently. Arc type lamps are also notoriously expensive; thus, frequent replacement can significantly increase laboratory costs. Furthermore, certain lamps are considered hazardous material, which complicates the disposal of the spent bulb.
Arc type lamps also feature a very small arc, which requires precision complex optics to properly enlarge the light source and fill the field of the microscope's optical system when a new lamp is installed. To align and focus the arc, a collector lens focus slider is used until one or two bow-tie shaped images (simulating a focused image of the arc and its mirror image) appear in the window. A lamp house mirror position slider is used to make the intensity of the two arc images approximately equal, and arc lamp horizontal and vertical sliders are utilized to overlay the two arc images into a single image. When the sliders have been adjusted to produce a small arc image in the center of the window, a collector lens focus slider is used to enlarge the arc image until it fills the entire window with a uniform field of illumination. As it can be seen, aligning and focusing an arc type lamp is complex and time-consuming.
A cheaper, more accurate, and more reliable alternative to an arc type lamp is a solid state light source, such as a light emitting diode (LED). LEDs are cheap, powerful, long-lasting sources of light. Moreover, a wide spectrum light source, such as an arc type lamp, provides a great deal of light that must be efficiently filtered out of the illumination to provide for an adequate signal to noise ratio. An LED's narrow bandwidth eliminates a great deal of the undesirable light, thereby reducing the requirements for filtration.
Nonetheless, there are several drawbacks to using LEDS within a fluorescence microscope. Each fluorescence stain fluoresces at a different wavelength; and therefore, each stain must be illuminated with a different monochromatic light source. Most users utilize several different stains. Thus, a microscope having a single LED would be severely limited in application.
The microscope may include multiple LEDs having different wavelengths, wherein the user may switch between LEDs to view differently stained specimens. However, each LED requires specific optical filters for filtering the light. Thus, the user would need to manually select and change the filters each time a new LED is installed within the microscope. Not only is this process time-consuming and cumbersome, it is susceptible to human error. Using an incorrect filter can cause damage to a user's eye. Thus, it is desired to provide a solid state fluorescence light system for a fluorescence microscope that can be used to view a variety of stains, that is easy to use, and that is not prone to human error.